The biosynthetic gene cluster for the antitumor drug bleomycin from ...

634 Chemistry & Biology 2000, Vol 7 No 8
Figure 7. A linear model for the BLM megasynthetase-templated assembly of the BLM peptide/polyketide/peptide aglycone from nine
amino acids and one acetate. Arrows with broken line indicate where biosynthetic intermediates were derailed. Three-letter amino acid
designations were used. NRPS and PKS domains are abbreviated as follows: A, adenylation; ACP, acyl carrier protein; AL, acyl CoA
ligase; AT, acyltransferase; C and CP, condensation; Cy, condensation/cyclization; KR, ketoreductase; KS, ketoacyl synthase; MT,
methyltransferase; Ox, oxidation; PCP, peptidyl carrier protein.
modules agree with the structure of the BLM peptide^
polyketide backbone. (3) The amino acid speci¢cities of
two NRPS modules were con¢rmed biochemically in vitro,
and the domain organization of the BlmIII and BlmIV
proteins, including the unusual Cy and Ox domains, concurs
with the unique bithiazole moiety of BLMs. (4) The
¢ve identi¢ed sugar biosynthesis genes, with their deduced
functions, correspond to the sugar moiety of
BLMs. (5) Fermentation of an S. lividans strain expressing
a part of the cloned gene cluster in the presence of exogenously
added BLM aglycones resulted in the production
of BLMs.
Most of the bacterial NRPS gene clusters characterized to
date are organized in operon-type structures, encoding
multimodular NRPS proteins with individual modules organized
along the chromosome in a linear order that parallels
the order of the amino acids in the resultant peptides,
i.e., following the `colinearity rule' for the NRPS-templated
assembly of peptides from amino acids [1^5]. Inspection
of the blm gene cluster (Figure 2B) showed that
the Blm NRPS and PKS modules apparently are not organized
according to the `colinearity rule' for BLM biosynthesis
(Figure 1B) (Exception to the `colinearity rule' was
also noted for the syringomycin synthetase from Pseudomonas
syringae [78] and for the exochelin synthetase from
M. smegmatis [75]. In fact, Grandi and co-workers have demonstrated
recently in B. subtilis that neither the operon-type
structure nor the physical linkage of individual modules is
essential for proper assembly and activity of the surfactin
NRPS megasynthetase [79].) Realizing that the BLM biosynthesis
cannot be rationalized according to the `colinearity
rule', we predicted the substrate speci¢cities of individual
NRPS modules according to the speci¢city conferring
codes for A domains [18] and deduced the extender unit of
the PKS module according to the signature motifs of the
AT domain [50]. Using the substrate speci¢city of individual
NRPS and PKS modules as a guide, we were able to
align the nine NRPS and one PKS modules to constitute
the Blm megasynthetase (Figure 7) according to our hybrid
NRPS^PKS^NRPS model for BLM biosynthesis (Figure
1B). Although its overall structure resembles that of known
NRPSs and PKSs, the Blm megasynthetase exhibits several
novel features. The latter is in fact expected since the
BLM backbone contains several unusual structural features,
such as the L-aminoalaninamide and the pyridimidine
moieties, both of which are unprecedented in peptide
biosynthesis.
On the basis of the BLM structure and the deduced functions
of individual NRPS and PKS domains, we propose
the following model for the Blm megasynthetase-templated
synthesis of BLMs. The individual modules are ¢rst
primed with amino acid or carboxylic acid precursors, the
substrate speci¢cities of which are determined by the A
domains of the NRPS or the AT domain of PKS modules,
respectively (Figure 7). We propose that the NRPS-4-activated
Ser is further dehydrated into dehydroalanine (Dha)
before its incorporation into the peptide product. Although
dehydroamino acids, such as Dha, are enamines and are
unlikely to be suf¢ciently stable, their stability could be
improved when they are linked covalently, via a thioester
bond, to the PCP domains and hence are sequestered by
the protein. Dha or dehydrothreonine (Dht), also known as
2,3-dehydroaminobutyric acid, are present in many peptides,
such as thiostrepton [83], microcystin [76], and syringomycin
[78]. Although it is yet to be determined if the
dehydration requires an additional enzyme, sequence analyses
of both the NRPS-4 module and the NRPS modules
for Dha in the microcystin cluster [76] and for Dht in the